A surprising discovery was made in the 1970s when researchers found that in many higher organisms, such as humans, only a small fraction of the genome encodes proteins, whereas the rest is seemingly junk DNA. In addition, the vast majority of genes have their protein-coding regions (exons) split up, separated by introns containing up to tens of thousands of nucleotides. We now know that non-protein-coding regions of the genome encode RNAs that contribute to gene regulation, catalysis and more, and the introns separating coding regions of genes are removed and the exons are joined together in a process called splicing. This critical step in gene expression allows for exquisitely fine-tund regulation and, through alternative splicing, allows a single gene to encode for more than one protein. Mistakes in this process can be lethal - it has been estimated that up to 60% of human genetic diseases involve defects in splicing. Splicing is executed by the spliceosome, a multi-megaDalton macromolecular machine whose function depends on the interplay between many protein and RNA components. Determining the roles of and interactions between these components is of central importance to understanding the process of splicing and, therefore, the molecular mechanisms of the many human diseases in which splicing is implicated. This proposal focuses on two proteins, Prp22 and Prp16, which facilitate structural rearrangements in the yeast spliceosome (which is very similar to that found in humans). These proteins function as RNA helicases in vitro, but it is not known how this helicase activity contributes to their function in the spliceosome. Specific Aim 1 involves studying the changes to pre-mRNA conformation induced by Prp22, separately examining its roles in the second catalytic step of splicing and in mRNA product release after the second step. Specific Aim 2 focuses on Prp16, which has been shown to play a proofreading role, triggering the discard of suboptimal pre- mRNA substrates prior to step 1 of catalysis. The approach will utilize the tools of single-molecule fluorescence resonance energy transfer (smFRET), which will provide the sensitivity to compare not only the pre-mRNA conformations present in different intermediate states in splicing, but also their dynamics. By introducing blocks in the splicing cycle using dominant negative mutations in Prp22 and Prp16, the changes in pre-mRNA conformation and dynamics induced by these proteins will be measured. An important facet of this work will be comparing the helicase/ATPase activities of Prp16 and Prp22 on model substrates in solution to their activities in the spliceosome. The knowledge provided by this project will be relevant to the understanding and treatment of diseases that involve defects in splicing. In addition, the project will provide important training in the areas of RNA biophysics, single-molecule spectroscopy, and scientific writing, presentation and mentoring.